Substrates for calcium-calmodulin-dependent phosphorylations were examined in both cytosolic and membrane-bound fractions of rat brain. Ca++-calmodulin-dependent protein kinase activity was partially purified using either DEAE-sephacryl of fluphenazine-Sepharose followed by calmodulin Sepharose. Substrates for phosphorylation differed markedly between preparations from DEAE-sephacryl and calmodulin-Sepharose. We also characterized ATP binding proteins in each preparation using the photoaffinity probe [alpha 32P]-8-azido-ATP. Numerous cytosolic proteins bound 8-azido-ATP when chromatographed over DEAE-sephacel. However, cytosol chromatographed over calmodulin-Sepharose exhibited only one protein band which bound ATP (M.W. = 49,000 daltons). The 49,000 dalton-ATP binding protein most likely represents the catalytic subunit of the protein kinase.